FMRP activity and control of Csw/SHP2 translation regulate MAPK-dependent synaptic transmission

Noonan syndrome (NS) and NS with multiple lentigines (NSML) cognitive dysfunction are linked to SH2 domain-containing protein tyrosine phosphatase-2 (SHP2) gain-of-function (GoF) and loss-of-function (LoF), respectively. In Drosophila disease models, we find both SHP2 mutations from human patients and corkscrew (csw) homolog LoF/GoF elevate glutamatergic transmission. Cell-targeted RNAi and neurotransmitter release analyses reveal a presynaptic requirement. Consistently, all mutants exhibit reduced synaptic depression during high-frequency stimulation. Both LoF and GoF mutants also show impaired synaptic plasticity, including reduced facilitation, augmentation, and post-tetanic potentiation. NS/NSML diseases are characterized by elevated MAPK/ERK signaling, and drugs suppressing this signaling restore normal neurotransmission in mutants. Fragile X syndrome (FXS) is likewise characterized by elevated MAPK/ERK signaling. Fragile X Mental Retardation Protein (FMRP) binds csw mRNA and neuronal Csw protein is elevated in Drosophila fragile X mental retardation 1 (dfmr1) nulls. Moreover, phosphorylated ERK (pERK) is increased in dfmr1 and csw null presynaptic boutons. We find presynaptic pERK activation in response to stimulation is reduced in dfmr1 and csw nulls. Trans-heterozygous csw/+; dfmr1/+ recapitulate elevated presynaptic pERK activation and function, showing FMRP and Csw/SHP2 act within the same signaling pathway. Thus, a FMRP and SHP2 MAPK/ERK regulative mechanism controls basal and activity-dependent neurotransmission strength.


Introduction
Noonan syndrome (NS) is an autosomal dominant genetic disorder caused by mutations in the mitogen-activated protein kinase (MAPK) pathway [1,2]. Missense mutations within the protein tyrosine phosphatase non-receptor type 11 (PTPN11) gene account for >50% of all disease cases [3]. In both patients and disease models, the MAPK pathway is hyperactivated by NS gain-of-function (GoF) mutations that disrupt the auto-inhibition mechanism between the a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 catalytic protein tyrosine phosphatase domain and N-Src homology-2 (SH2) domainAU : Anabbreviatio of the PTPN11 encoded SH2 domain-containing protein tyrosine phosphatase-2 (SHP2; [4,5]). In the NS with multiple lentigines (NSML) disease state, PTPN11 loss-of-function (LoF) mutations decrease protein tyrosine phosphatase domain catalytic activity, but the mutants nevertheless maintain a more persistently active enzyme state with temporally inappropriate SHP2 function, causing elevated MAPK pathway hyperactivation similar to the GoF disease condition [6]. Consequently, NS and NSML patients share a great many symptoms associated with elevated MAPK signaling, including cognitive dysfunction (approximately 30% of cases) as well as long-term memory (LTM) impairments [7,8]. The Drosophila NS (GoF) and NSML (LoF) disease models from mutation of the corkscrew (csw) homolog likewise both increase MAPK activation, with GoF and LoF also phenocopying each other [9,10]. Drosophila LTM training generates repetitive waves of csw-dependent neural MAPK activation, with the LTM spacing effect misregulated by csw manipulations [11]. PTPN11 GoF and LoF mutations from human patients transgenically introduced into the Drosophila model provide a powerful new means to compare with csw GoF and LoF mutants in the dissection of conserved neuronal requirements [12].
Fragile X syndrome (FXS) is similarly well characterized by hyperactivated MAPK signaling within neurons [13], and the causal Fragile X Mental Retardation Protein (FMRP) RNA-binding translational regulator is proposed to directly bind PTPN11/SHP2 mRNA [14,15]. FMRP also binds many other neuronal transcripts [16] and could interact with SHP2 in multiple ways to coregulate the MAPK pathway. Moreover, like the NS and NSML disease states, FXS is likewise a cognitive disorder and the leading heritable cause of intellectual disability [16]. Like NS and NSML, the Drosophila FXS disease model also manifests strongly impaired LTM consolidation [17,18]. Mechanistically, MAPK signaling is well known to modulate glutamatergic synaptic neurotransmission strength via the control of presynaptic vesicle trafficking dynamics and glutamate neurotransmitter release probability [19]. Consistently, FMRP is also well characterized to regulate glutamatergic synaptic neurotransmission, including presynaptic release properties and activity-dependent functional plasticity [20]. Importantly, treatment with the MAPK inhibitor Lovastatin corrects hippocampal hyperexcitability in the mouse FXS disease model and ameliorates behavioral symptoms in human FXS patients [21,22]. In the Drosophila FXS disease model, dfmr1 null mutants show elevated presynaptic glutamate release underlying increased neurotransmission strength [17], as well as activity-dependent hyperexcitability and cyclic increases in glutamate release during sustained high-frequency stimulation trains [23]. Based on this broad foundation, we hypothesized that FMRP regulates PTPN11 (SHP2)/ Csw translation to modulate presynaptic MAPK signaling, which, in turn, controls presynaptic glutamate release probability to determine both basal neurotransmission strength and activitydependent synaptic plasticity.
To investigate this hypothesis, we utilized the Drosophila neuromuscular junction (NMJ) glutamatergic model synapse with the combined use of NS, NSML, and FXS disease models. We first tested both LoF and GoF conditions in both (1) csw mutants and (2) transgenic human PTPN11 lines. In two-electrode voltage-clamp (TEVC) electrophysiological recordings, all of these mutant conditions elevate synaptic transmission. We next employed cell-targeted RNAi and spontaneous miniature excitatory junction current (mEJC) recordings to find Csw/ SHP2 specifically inhibits presynaptic glutamate release probability. We next tested activitydependent synaptic transmission using high-frequency stimulation (HFS) depression assays to show that the mutants display heightened transmission resiliency, consistent with elevated presynaptic function. We discovered that both LoF and GoF mutations impair presynaptic plasticity, with decreased short-term facilitation, maintained augmentation and post-tetanic potentiation (PTP), supporting altered presynaptic function. Consistent with elevated MAPK signaling in NS, NSML, and FXS disease models, feeding with MAPK-inhibiting drugs (Trametinib and Vorinostat) corrects synaptic transmission strength in mutants. As predicted, we found that FMRP binds csw mRNA and that FMRP loss increases Csw protein levels. Both dfmr1 and csw nulls display elevated phosphorylated ERK (pERK) in presynaptic boutons. Importantly, trans-heterozygous double mutants (csw/+; dfmr1/+) exhibit presynaptic MAPK signaling and neurotransmitter release phenotypes, indicating FMRP and Csw/SHP2 operate to control MAPK/ERK signaling and synaptic function. These discoveries link previously unconnected disease states NS, NSML, and FXS via a presynaptic MAPK/ERK regulative mechanism controlling glutamatergic transmission.

Corkscrew/PTPN11 controls presynaptic transmission by altering glutamate release probability
Our next objective was to determine where Corkscrew acts to mediate synaptic changes in neurotransmission strength. To test requirements, we knocked down csw expression through RNA interference (RNAi) driven in the different cells contributing to the NMJ, including the presynaptic motor neuron and postsynaptic muscle [29]. We used targeted transgenic RNAi against csw (BDSC 33619; [30]) to test each cell-specific function. This line is from the Harvard Transgenic RNAi Project (TRiP), which provides a background control stock (BDSC 36303) containing all components except the UAS-RNAi [31]. To test RNAi efficacy and replication of csw 5 null phenotypes, we first used the ubiquitous daughterless UH1-Gal4 driver. To separate cellular requirements, we used neuronal elav-Gal4 and muscle 24B-Gal4-specific drivers, each compared to their respective driver alone transgenic controls. With each RNAi knockdown, we once again utilized TEVC recordings of evoked EJC neurotransmission to measure synaptic strength. To further test csw functional roles, we analyzed spontaneous release events by assessing changes in both frequency and amplitude with miniature EJC (mEJC) recordings [28]. Changes in the mEJC frequency are correlated with alterations in presynaptic fusion probability, whereas changes in mEJC amplitudes indicate differential postsynaptic glutamate receptor function or altered vesicle size [32,33]. We made continuous mEJC recordings collected over 2 minutes using a gap-free configuration filtered at 10 kHz [28]. Each data point corresponds to the mean mEJC frequency and amplitude of all the recorded release events. Representative recordings and quantified results are shown in Fig 2. The ubiquitous transgenic driver control (UH1-Gal4/TRiP BDSC 36303 control) exhibits neurotransmission indistinguishable from the w 1118 genetic background control (Fig 2A, left). Ubiquitous csw knockdown (UH1>csw RNAi) causes elevated neurotransmission closely consistent with the csw 5 null mutant (Fig 2A, second from left), demonstrating RNAi efficacy as well as null phenocopy (compare to Fig 1A, left). The quantified EJC measurements show UH1>csw RNAi (233.20 ± 17.45 nA, n = 10) to be strongly elevated compared to controls (152.30 ± 15.65 nA, n = 10), which is a significant increase (p = 0.003, two-sided t test; Fig 2B). The neuronal driver control (elav-Gal4/TRiP BDSC 36303 control) compared to neuronalspecific knockdown (elav>csw RNAi) also shows strong replication of the csw 5 null elevated transmission, indicating a primary csw requirement in the presynaptic neuron (Fig 2A, middle pair). Quantified measurements show elav>csw RNAi EJC amplitude (239.70 ± 19.45 nA, n = 10) also strongly increased compared with the elav-Gal4/TRiP driver controls (159.90 ± 9.68 nA, n = 12), which is significant (p = 0.001, two-sided t test; Fig 2B, middle). In contrast, targeted muscle RNAi knockdown (24B>csw RNAi) does not cause any change in evoked neurotransmission compared to the muscle driver control alone (24B-Gal4/TRiP BDSC 36303; Fig  2A, right pair), signifying that postsynaptic Csw does not detectably change synaptic function. When quantified, 24B-Gal4/TRiP (156.50 ± 11.41 nA, n = 10) is comparable to 24B>csw RNAi (170.30 ± 11.24 nA, n = 11), with no significant change in amplitude (p = 0.401, twosided t test; Fig 2B, right). These findings indicate a primary csw requirement in presynaptic neurons regulating glutamate neurotransmitter release.

Corkscrew/PTPN11 regulates high-frequency stimulation synaptic depression
To further investigate how csw/PTPN11 affects presynaptic neurotransmission strength, we stimulated at a heightened frequency that has been shown to cause synaptic depression over a time course of several minutes [34][35][36]. Synaptic depression occurs when HFS causes synaptic vesicles to be released at a faster rate than they can be replenished in presynaptic boutons [34,37]. Based on published HFS protocols for the Drosophila NMJ [34,36,38], we compared the genetic background control (w 1118 ), csw null LoF mutant (csw 5 ), and patient-derived PTPN11 N308D GoF mutant (elav-Gal4>PTPN11 N308D ) with a HFS paradigm. To determine the baseline EJC amplitudes, we first stimulated for 1 minute under basal conditions (0.5 ms suprathreshold stimuli at 0.2 Hz in 1.0 mM external [Ca 2+ ]). We then stimulated at 100X greater frequency (20 Hz) for 5 minutes while continuously recording EJC responses. This sustained HFS train causes progressively decreased neurotransmission over time (depression). HFS transmission was quantified to analyze the synaptic vesicle readily releasable pool (RRP) and paired-pulse ratio (PPR) release probability. Representative HFS recordings and quantified results are shown in Figs 3 and S7.
During HFS, w 1118 controls exhibit a steady decrease in EJC amplitudes throughout the train (Fig 3A, top). The PTPN11 N308D GoF mutants and csw 5 LoF nulls show stronger maintained EJC amplitudes over time and prolonged resistance to depression (Figs 3A and S7A). RRP size was calculated by dividing the cumulative EJCs during the first 100 responses by mean mEJC amplitudes [39]. There is a sustained elevated response in both LoF and GoF mutants ( Fig 3B). When compared with nonlinear regression and extra sum-of-squares, the stimulation train profiles are significantly greater for both LoF (p < 0.0001, F (2,1296) = 1064) and GoF (p < 0.0001, F (2,1996) = 705.5; Fig 3B) mutants, indicating increased resiliency to depression. The RRP size of csw 5 nulls is significantly increased compared to w 1118 background controls (p = 0.001, two-sided t test; Fig

Elevated Corkscrew/PTPN11 synaptic transmission corrected with pERK inhibitors
NS and NSML phenotypes are hypothesized to converge due to both LoF/GoF disease states exhibiting constitutively elevated MAPK/ERK signaling [10]. Similarly, we hypothesize the mutant LoF/GoF neurotransmission elevation from heightened glutamate release also occurs downstream of elevated presynaptic MAPK/ERK signaling. To test this hypothesis, we used MAPK/ERK inhibitors (Trametinib and Vorinostat) to assay effects on glutamatergic synaptic function. Trametinib binds and inhibits MEK1/2 [45], resulting in a direct inhibition of MAPK/ERK signaling [12]. Vorinostat acts as a HDAC inhibitor to also inhibit MAPK/ERK signaling [12,46]. Recent work using the PTPN11 mutations from human patients has highlighted these two drugs as possible treatments for a variety of different NS/NSML mutations [12]. Both drugs are thus interesting not only for their ability to test elevated MAPK/ERK signaling upstream of neurotransmission, but also as possible future treatment avenues. We fed both drugs and then analyzed changes in EJC amplitudes using TEVC recording. For each

FMRP binds csw mRNA to suppress Csw protein expression upstream of MAPK/ERK signaling
The FMRP negative translational regulator is well known to inhibit MAPK/ERK signaling in the regulation of synaptic function [13]. Moreover, high-throughput RNA sequencing from isolated crosslinking immunoprecipitation shows FMRP binds csw homolog PTPN11/SHP2 mRNA [14]. Therefore, we hypothesized FMRP binds csw mRNA to negatively regulate translation upstream of MAPK/ERK signaling. To test this hypothesis, we first performed RNAimmunoprecipitation (RIP) studies with tagged FMRP::YFP from larval lysates using magnetic GFP-trap beads [47,48]. We used Tubby::GFP lysates as the RIP negative control, with α-tubulin (FMRP does not bind) as the internal negative control, and futsch/MAP1B (known FMRP target) as the internal positive control [17]. Immunoprecipitated mRNAs were reverse transcribed and tested with specific primers on 2% agarose gels. We next used western blots from larval ventral nerve cord (VNC)/brain lysates to test neuronal Csw protein levels with a characterized anti-Csw antibody [9]. Antibody specificity was confirmed with the csw 5 null and protein levels compared between the genetic background control (w 1118 ) and FXS disease model (dfmr1 null mutants). To compare neuronal Csw protein levels in these different genotypes, we normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a housekeeping gene that we confirmed is not regulated by Csw. Normalized quantification was done to compare neuronal Csw protein levels in the w 1118 controls, csw 5 null mutants, and dfmr1 50M null mutants. Representative RIP gels, western blots, and western blot quantified data are shown in Fig 6. For the RIP analyses, csw, futsch, and α-tubulin mRNA bands are all present in both Tubby::GFP control and FMRP::YFP input lysates (Fig 6A). Immunoprecipitation pulls down csw mRNA from the FMRP::YFP third instar lysates, with no binding in the Tubby::GFP control ( Fig 6A). Additionally, the positive control futsch mRNA is pulled down, but there is no detectable negative control α-tubulin mRNA. These results indicate FMRP binds csw mRNA, with the controls confirming binding interaction specificity. Based on this and above findings, we hypothesized FMRP partly inhibits NMJ synaptic transmission by suppressing Csw translation in neurons to decrease MAPK/ERK signaling. To test this hypothesis, western blot analyses were done to test Csw protein levels in larval brain/VNC lysates from controls (w 1118 ), csw 5 , and dfmr1 50M null mutants. At the predicted molecular weight (100 kDa), there is a clear Csw band present in controls (Fig 6B). This band is undetectable in csw 5 nulls, demonstrating specificity ( Fig 6B). In the FXS disease model, there are clearly and consistently increased Csw protein levels in dfmr1 null mutants (Fig 6B). Quantified comparisons normalized to GAPDH (p < 0.0001, ANOVA) show an increase in Csw levels in dfmr1 nulls (1.55 ± 0.13) compared to controls (0.99 ± 0.029), which reveals a highly significant increase in the FXS disease model (p = 0.0008, Tukey's, Fig 6C). There is slight background in csw 5 (0.23 ± 0.06), which is very significantly decreased from controls (p < 0.0001, Tukey's) and dfmr1 mutants (p < 0.0001, Tukey's; Fig 6C). Thus, dfmr1 nulls have a strong increase in Csw levels in the larval neurons. Taken together, these findings show FMRP binds csw mRNA to negatively regulate Csw protein levels. We hypothesized this interaction negatively regulates presynaptic MAPK/ERK signaling.

FMRP and Csw interact to inhibit presynaptic MAPK/ERK signaling and neurotransmission
We next set forth to test MAPK/ERK signaling within presynaptic boutons in order to begin investigating how FMRP and Csw interact to control presynaptic transmission. Elevated presynaptic pERK is well known to positively regulate neurotransmitter release function [49]. Based on this known role and our above studies, we hypothesized locally elevated pERK levels should occur in both csw and dfmr1 null synaptic boutons. To test this hypothesis, we assayed NMJ terminals double-labeled with anti-pERK [50] and anti-horseradish peroxidase (HRP) to mark presynaptic bouton membranes. Using HRP to delineate presynaptic boutons, we measured pERK fluorescence intensity normalized to the genetic background control (w 1118 ). Presynaptic pERK signaling is activity-dependent [51,52]. To test this function, we compared presynaptic pERK levels in the basal resting condition to stimulation with acute (10 minute) high [K + ] depolarization (90 mM; [53,54]) in w 1118 control, dfmr1 50M null mutant, and csw 5 null mutant. We hypothesized that FMRP and Csw interact to inhibit presynaptic pERK signaling-dependent transmission strength. To test this hypothesis, we assayed the double transheterozygous csw 5 /+; dfmr1 50M /+ mutant compared to both single heterozygous mutants alone [28]. We first used TEVC recordings to measure stimulation evoked EJC responses and spontaneous mEJC release events. We then used pERK/HRP double-labeled imaging to Activated pERK is weakly detectable at control synapses under basal resting conditions ( Fig  6D, top). In w 1118 controls, pERK is localized at relatively higher levels in the presynaptic boutons, with lower levels of signaling in the adjacent muscle nuclei and very low sporadic levels throughout the muscle. Given the consistent presynaptic phenotypes above, we focused analyses on pERK signaling within presynaptic boutons. Compared to controls, both csw and dfmr1 null mutants display consistently elevated pERK levels within the presynaptic boutons (Fig 6D,  top), but with similar levels of pERK fluorescence in muscle compared to the controls. Similar results occur in PTPN11 human patient mutants compared to driver controls (S9A Fig), with elevated pERK levels in all conditions (S9B Fig). This increased presynaptic pERK signaling and lack of postsynaptic changes is consistent with presynaptic perturbations in both csw and dfmr1 null mutants. Quantification of the normalized pERK fluorescent intensity within the HRP-delineated presynaptic boutons shows very highly elevated levels in both the csw (1.85 ± 0.25, n = 15) and dfmr1 (1.58 ± 0.13, n = 18) null mutants compared to controls (1.0 ± 0.12, n = 24), which is a significant increase (p = 0.001, one-way ANOVA; Fig 6E). When compared individually, there is no significant difference between dfmr1 and csw mutants (p = 0.526, Tukey's), showing both csw (p = 0.001, Tukey's) and dfmr1 (p = 0.024, Tukey's; Fig 6E) nulls increase pERK signaling to a similar degree compared to controls. This elevated presynaptic pERK in both disease models fits our hypothesis that elevated MAPK/ ERK signaling causes the increased presynaptic transmission in both disease models. Given the above changes in activity-dependent presynaptic function in csw null mutants, we next wanted to test whether pERK levels are dynamic and change with a stimulation challenge, and whether activity-dependent impairments occur in the two disease models.
https://doi.org/10.1371/journal.pbio.3001969.g006 nulls display only a trending elevation in stimulated pERK levels, without a significant increase from rest (p = 0.083, two-sided t test; Fig 6G). Likewise, dfmr1 nulls display a reduced activitydependent increase in stimulated presynaptic pERK levels compared to the basal condition, albeit still significant (p = 0.014, two-sided t test; Fig 6G). We conclude that the basal elevation in pERK levels in both disease models blunts further activation in response to stimulation. This activity-dependent defect correlates with the above impaired functional neurotransmission dynamics in response to stimulation. Based on the perturbed presynaptic pERK signaling in csw and dfmr1 nulls, we hypothesized FMRP and Csw interact to inhibit synaptic MAPK/ ERK signaling and transmission.
We therefore directly tested for this mechanism with csw/+; dfmr1/+ trans-heterozygotes. In TEVC recordings, these trans-heterozygotes show elevated neurotransmission compared to w 1118 controls and both of the single heterozygotes (S10A

Discussion
MAPK is well known to regulate activity-dependent signal transduction and synaptic plasticity within the nervous system [55]. Four MAPK families have been characterized, including extracellular signal-regulated kinase 1/2 (ERK1/2), ERK5, p38 MAPK, and the c-Jun N-terminal kinase (JNK; [56]). These families are activated similarly through an evolutionarily conserved cascade involving initial activation of GTPases (Ras/Rac) and a subsequent three-tiered protein kinase signaling system [57]. The best-characterized MAPK pathway, ERK1/2, has been extensively investigated within the nervous system, where ERK activation is very tightly regulated. Numerous neurological disease states display elevated ERK activity, including FXS, NS, and NSML, as well as neurodegenerative diseases such as Alzheimer's and Parkinson's disease [10,13,58]. Many studies have linked such elevated ERK signaling to cognitive deficits, particularly impairment of LTM consolidation. LTM requires spaced learning sessions during which ERK is activated and then decays in a temporal cycle. In Drosophila PTPN11/SHP2 homolog csw mutants, this ERK activation timing cycle is perturbed and LTM is disrupted [11].
Moreover, one of the targets of FMRP, a negative translational regulator, is PTPN11/SHP2 mRNA [14], suggesting a potential link between the FXS and NS/NSML disease states. Based on the common ERK signaling up-regulation in these disorders, we hypothesized FMRP regulates Csw translation to modulate synaptic ERK levels to control neurotransmission strength and functional plasticity.
This hypothesis provides the first proposed mechanistic connection between NS, NSML, and FXS disease conditions, through an ERK phosphorylation (pERK) signaling defect in presynaptic boutons. pERK is known to activate presynaptic function, with short-term roles in the control of neurotransmission strength and activity-dependent plasticity [49,59], and longer-term nuclear translocation roles [57]. In the Drosophila NS/NSML disease models of csw LoF and GoF, we began with synaptic transmission assays at the NMJ glutamatergic synapse [32]. We also tested human patient PTPN11/SHP2 mutations to confirm functional requirements [12]. Our work reveals that all LoF/GoF mutations elevate neurotransmission strength, indicating that Csw/SHP2 is involved in inhibiting glutamatergic signaling. Consistently, previous Drosophila NS and NSML model studies also show that LoF and GoF mutations phenocopy one another, with a correlation to hyperactivated pERK signal transduction in both conditions [9,10]. Moreover, the Drosophila FXS disease model similarly increases NMJ glutamatergic synaptic transmission [17], consistent with the FMRP mechanistic intersection. Localized pERK signaling occurs on both pre-and postsynaptic sides [60,61], so we next used cell-targeted csw RNAi and measured spontaneous vesicle fusion events to separate these requirements. Our work reveals Csw/SHP2 has only a neuronal role in the regulation of presynaptic transmission. There is no detectable postsynaptic function. This new presynaptic Csw/SHP2 role is consistent with the abundant evidence for both MAPK/ERK and FMRP involvement in modulating glutamatergic release mechanisms.
Presynaptic vesicle fusion is a major determinant of neurotransmission strength, maintained functional resilience during strong demand, and activity-dependent plasticity [62]. HFS trains cause the transient activation of pERK signaling in presynaptic terminals [51], correlating with increased vesicle fusion. To test if Csw/SHP2 similarly regulates glutamate release, we performed HFS synaptic depression assays to discover that all mutants have increased transmission resiliency under conditions of heightened demand [34], with elevated glutamate release from presynaptic boutons. This role is consistent with activity-dependent presynaptic MAPK/ERK signaling driving greater presynaptic glutamate release by modulating the accessible number of synaptic vesicles available for fusion in the RRP [19]. Importantly, the mouse FXS disease model displays similar decreased short-term depression due to enhanced presynaptic glutamate release, also via up-regulation of the RRP without a change in PPR fusion [20]. The MAPK/ERK-dependent phosphorylation of presynaptic targets is likewise known to increase short-term plasticity, and blockade of this signaling process has been shown to strongly impair facilitation, maintained augmentation, and PTP [51,63]. Our results show that all three forms of synaptic plasticity are impaired in csw null and PTPN11 N308D GoF animals, which both show decreased facilitation, augmentation, and PTP, consistent with other LoF/ GoF phenocopy. We hypothesize that these plasticity defects correlate to the already increased basal transmission levels that cause a decrease in range for enhancement from presynaptic pERK activation, leading to a "ceiling effect" on presynaptic function. This predicts neurotransmission defects are linked to causal changes in presynaptic MAPK/ERK signaling.
Both NS and NSML disease states exhibit elevated MAPK/ERK signaling [10], but there is heterogeneity in pERK activation levels and multiple pathways involved [12]. To confirm the neurotransmission increase is due to elevated MAPK/ERK signaling, we inhibited this pathway with both Trametinib and Vorinostat, two drugs well characterized to decrease pERK signaling [46,64]. With drug treatments, the elevated neurotransmission in csw and PTPN11 mutants is restored to levels comparable to control animals, indicating that the elevated MAPK/ERK signaling is responsible for the heightened presynaptic function. This test does not rule out the possibility of other disrupted signaling pathways that may influence MAPK/ ERK signaling, but does prove MAPK/ERK signaling is the cause of the elevated neurotransmission. The next task was to explore the new activity-dependent mechanism controlling this presynaptic Csw/SHP2 function. As previously discussed, NS, NSML, and FXS models/ patients all display striking similarities in up-regulated MAPK/ERK signaling, synaptic phenotypes, and LTM impairments [17,18,20]. Moreover, RNA-binding FMRP is well characterized as an activity-dependent negative translational regulator of presynaptic mRNA targets [65]. Consistently, we find that Drosophila FMRP binds csw mRNA, as suggested in a mouse FMRP screen indicating PTPN11/SHP2 binding [14]. Additionally, we find neuronal Csw protein levels are elevated in the FXS disease model (dfmr1 null), consistent with the predicted FMRP translational repression [66]. Finally, we find that presynaptic pERK signaling is increased in both dfmr1 and csw null mutants and that normal activity-dependent elevation in pERK signaling is impaired in both disease model conditions. The pERK enhancement levels are slightly different, but this to likely due to the relative effect of the two nulls on pERK signaling. The heightened basal presynaptic pERK signaling and repressed activity-dependent pERK signaling suggests that FMRP and Csw interact to modulate presynaptic glutamatergic neurotransmission.
One genetic test for pathway interaction employs nonallelic noncomplementation [67], which demonstrates that the two gene products operate within a common mechanism, in this case, the up-regulation of MAPK signaling [28]. Both dfmr1 and csw null mutants display elevated presynaptic neurotransmission with an increased probability of presynaptic glutamate release [17], and trans-heterozygous dfmr1/+; csw/+ double mutants recapitulate both functional phenotypes. Importantly, both the dfmr1 and csw 5 single heterozygous mutants do not display any phenotypes, despite the NSML autosomal dominant disease state. Similarly, Csw/ PTPN11 overexpression does not cause any phenotypes, suggesting a change in the FXS background causes the elevated MAPK/ERK presynaptic signaling. These genetic tests indicate that FMRP and Csw/SHP2 act together to inhibit pERK signaling and presynaptic glutamate release. We propose the mechanism of mRNA-binding FMRP acting canonically as a negative translational regulator of Csw/SHP2 expression [68]. Both the dfmr1 and csw null mutants display elevated MAPK/ERK signaling as indicated by pERK production [56], and we demonstrate here pERK elevation in presynaptic boutons. Consistent with a common mechanism, trans-heterozygous csw/+; dfmr1/+ mutants recapitulate this heightened presynaptic pERK signaling. We propose the mechanism of FMRP working through Csw/SHP2 phosphatase enzymatic activity to inhibit presynaptic pERK production. Given that MAPK/ERK signaling is well established to modulate presynaptic glutamatergic release [49], we suggest heightened presynaptic pERK signaling causes elevated glutamate release probability. We demonstrate this causal link with pharmacological treatments that block pERK production [45], which act to restore normal glutamatergic synaptic signaling in the disease model animals.
In conclusion, we note that there are important differences between FXS and NS/NSML disease models. Previous FXS model work has shown increased NMJ architecture and mEJC amplitudes in dfmr1 nulls [17], which are absent in NS/NSML model csw/PTPN11 mutants. FXS is a very complex disease state with many proteins misregulated [17], and there was never an expectation that all FXS phenotypes would be recapitulated in csw/PTPN11 mutants, especially for the unrelated postsynaptic changes. Nevertheless, the presynaptic parallels are striking. The mouse FXS model exhibits decreased short-term depression with no change in PPR, but an increase in RRP [20], matching the Drosophila results shown here. Interestingly, these phenotypes match closer than mouse H-ras G12V mutants with increased pERK signaling, which exhibit enhanced short-term synaptic plasticity [19], compared to the depressed plasticity shown here. Thus, although both basal transmission strength and functional plasticity properties are dependent on presynaptic MAPK/ERK signaling, there are likely other intersecting regulatory pathways. Moreover, FMRP and Csw/SHP2 could interact via multiple different mechanisms to regulate presynaptic MAPK/ERK signaling, and the elevated neurotransmission in the disease state models may not be completely dependent on presynaptic MAPK/ERK signaling. In the FXS model, Csw/SHP2 is both up-regulated and hyperactivated, and the mechanism of this activation is unknown. One possibility is decreased MAPK/ ERK negative regulation, via other factors like Neurofibromin-1, which could further increase MAPK/ERK signaling [69,70]. Another possibility is that neuronal activity up-regulates and then activates Csw/SHP2 via two parallel mechanisms to increase MAPK/ERK signaling [71,72]. We have previously uncovered several other genetic mutants that likewise elevate neurotransmission and depress short-term plasticity [28,[73][74][75], which are also candidates for furthering our understanding in future studies. The possibility for a more extensive interactive molecular network is exciting, but it can currently only be concluded that FMRP and Csw/ SHP2 both control MAPK/ERK signaling and modulate neurotransmission. This presynaptic mechanism connects the previously unlinked disorders of NS, NSML, and FXS, suggesting common therapeutic targets and new treatment avenues.

Drosophila genetics
All the Drosophila stocks were reared on standard cornmeal/agar/molasses food at 25˚C within 12-hour light/dark cycling incubators. All animals were reared to the wandering third instar stage for all experiments, with all genotypes and RNAi lines confirmed with a combination of transgenically marked balancer chromosomes, western blots, and sequencing. Due to the corkscrew gene being on the X chromosome, all experiments utilizing csw 5 mutants were conducted using males only, whereas all the trans-heterozygous experiments were done using females only. All the other experiments were done on both of the sexes (males and females together). The two genetic background controls were w 1118 and the TRiP RNAi third chromosome background control [31]. The dfmr1 50M null mutant [17], csw 5 null mutant [24], and the transgenic lines UAS-csw WT and UAS-csw RNAi [25,30] are all available from the Drosophila Bloomington Stock Center (BDSC; Indiana University, Bloomington, IN, USA). The UAS-csw A72S line [9] was obtained as a kind gift from Dr. Mario Rafael Pagani (Department of Physiology and Biophysics, School of Medicine, National Scientific and Technical Research Council, University of Buenos Aires, Buenos Aires, Argentina). All patient-derived UAS-PTPN11 mutant lines [12] were obtained as a kind gift from Dr. Tirtha Das (Department of Cell, Developmental, and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY, USA). Transgenic studies were performed with neural-specific elav-Gal4 [76], muscle-specific 24B-Gal4 [77], and ubiquitous daughterless UH1-Gal4 [78] driver lines, all obtained from BDSC. The genetic and transgenic lines used in this study are summarized below in Table 1:

Synaptic electrophysiology
Wandering third instar dissections and TEVC recordings were done at 18˚C in physiological saline (in mM): 128 NaCl, 2 KCl, 4 MgCl 2 , 1.0 CaCl 2 , 70 sucrose, and 5 HEPES (pH 7.2). Staged larvae were dissected longitudinally along the dorsal midline, the internal organs removed, and the body walls glued down (Vetbond, 3M). Peripheral motor nerves were cut at the base of the VNC. Dissected preparations were imaged with a Zeiss 40× water-immersion objective on a Zeiss Axioskop microscope. Muscle 6 in abdominal segments 3 to 4 was impaled with two intracellular electrodes (1 mm outer diameter borosilicate capillaries; World Precision Instruments, 1B100F-4) of approximately 15 MO resistance when filled with 3M KCl. The muscles were clamped at −60 mV using an Axoclamp-2B amplifier (Axon Instruments). For evoked EJC recordings, the motor nerve was stimulated with a fire-polished suction electrode using 0.5 ms suprathreshold voltage stimuli at 0.2 Hz from a Grass S88 stimulator. Nerve stimulation-evoked EJC recordings were filtered at 2 kHz. To quantify EJC amplitude, 10 consecutive traces were averaged, and the average peak value recorded. Spontaneous mEJC recordings were made in continuous 2-minute sessions and low-pass filtered at 200 Hz. Synaptic depression experiments were performed using the above EJC recording protocol for 1 minute to establish baseline, followed by a 20-Hz HFS train for 5 minutes at the same suprathreshold voltage. RRP size was estimated by dividing the cumulative EJC amplitudes during the first 100 responses to 20 Hz stimulation by the mean mEJC amplitudes. Due to these analyses being at 20 Hz, RRP size is likely underestimated. All synaptic plasticity experiments were performed in 0.2 mM Ca 2+ using 10 Hz stimulation trains for 1 minute, followed by 0.2 Hz recordings. All EJC responses within a 1-second bin were averaged, and the average value normalized to the basal EJC amplitude for each animal. Clampex 9.0 was used for all data acquisition, and Clampfit 10.6 was used for all data analyses (Axon Instruments).

Drug treatments
Two drugs known to inhibit pERK production (Trametinib and Vorinostat) were used by feeding as published previously [12,45,46]. Both Trametinib (Cell Signaling, 62206S) and Vorinostat (Cell Signaling, 12520S) were dissolved in dimethylsulfoxide (DMSO; Fisher, 67-68-5) at 15 mM and 20 mM, respectively, to create stock solutions. Both drugs were then added to Drosophila food yeast paste and in the standard cornmeal/agar/molasses food in the final concentrations of 0.5 mM (Trametinib) and 1 mM (Vorinostat). Drosophila were induced to lay eggs on selection apple juice plates with drugged yeast paste food. Hatching first instars were selected and placed in standard vials containing Trametinib, Vorinostat, or control food with DMSO only. Larvae were reared in a 12-hour light/dark cycling incubators at 25˚C and then collected as wandering third instars for TEVC studies.

RNA immunoprecipitation
Wandering third instars (20 larvae) of each genotype (UH1>FMRP-YFP or Tubulin-GFP) were homogenized in 200 μL of RNase-free lysis buffer (20 mM HEPES, 100 mM NaCl, 2.5 mM EDTA, 0.05% (v/v) Triton X-100, 5% (v/v) glycerol) with 1% β-mercaptoethanol 1× protease inhibitor cocktail (complete mini EDTA-free Tablets, Sigma, 11836170001) and 400U RNase inhibitor (Applied Biosystems, N8080119). The supernatant was collected and diluted to 300 μL to reduce nonspecific binding. Next, the samples were incubated with GFP-trap coupled magnetic agarose beads (Chromotek, GTMA20) for 3 hours at 4˚C. The bound beads were washed with lysis buffer (3X, 10 minutes). The bound RNA was purified by incubating the bead-protein-RNA conjugates with a 500-μL TRIzol and chloroform mixture (Ambion, 15596026) for 10 minutes at RT, followed by centrifugation. To precipitate RNA, glycogen (1 μL) and 2-propenol (250 μL) were added to the isolated aqueous layer. Finally, the precipitated RNA was reverse transcribed into single-strand cDNA using the SuperScript VILO cDNA synthesis kit (Thermo Fisher, 11754050) and then subjected to primer-specific PCR, with 2% agarose gels used to analyze the PCR products. All primers used in this study are summarized above in Table 2.